In the area of enzyme design and engineering, DNA restriction endonucleases (REs) are of particular interest and present compelling challenges. Restriction enzymes recognize short defined DNA sequences and cleave the DNA sugar-phosphate backbone with considerable efficiency. These enzymes have been studied for over half a century and have given rise to the era of recombinant biotechnology in the past few decades. Yet, their structural features and propensities that lend their functional ability to recognize short DNA sequences in the genome have yet to be understood at predictive proficiency. The general goal of this research is to develop a computational tool to enable study and reengineering of DNA restriction enzymes. The package will include the following components: 1) nucleic acid recognition site perturbation tools, which will allow sequence recognition, structural modeling and visualization of the nucleic acid recognition site;2) a biological focusing algorithm which will enumerate and focus on those scaffolds and interfacial pockets (IPs) that have potential for design, as demonstrated by evolutionarily favorable features;and 3) a DNA endonuclease library integration and querying interface which will streamline the cataloging, retrieval, use, and dissemination of analyses performed using the above tools and algorithm. These computational tools, once developed, will help launch systemic research into the fundamentals of structural biology and engineering of restriction endonucleases. The development of such innovative biomedical computing technologies will be facilitated by the research group's strength in high-performance molecular modeling and optimization as well as high-throughput gene synthesis, protein expression, and screening technologies. This research will eventually translate into custom-designed endonucleases for sequence-specific biomolecular probes or clinical therapeutic in further investigations or treatments.